This invention relates to gene expression.
The organisms grouped as Nocardioforms in Bergey's Manual of Systematic Bacteriology (Williams and Wilkins, 1986, pp. 1458-1481) include nine genera which are close phylogenetically to Corynebacterium, Athrobacter and Mycobacterium. Members of the Nocardioforms including the genera Nocardia and Rhodococcus exhibit a wide range of useful metabolic activities including the assimilation of unusual compounds such as alkanes and aromatic hydrocarbons; the degradation of lignin, detergents and pesticides; the production of enzymes useful in xenobiotic transformations; and the biosynthesis of antibiotics, amino acids and biosurfactants. In particular, this group of bacteria have been exploited for their production of important steroid modifying enzymes, including cholesterol esterase and cholesterol oxidase, which are used in diagnostic assays to determine cholesterol levels in blood and serum.
The capacity to degrade cholesterol is widespread among microorganisms other than the Nocardioforms. For example, cholesterol oxidases have also been isolated from species of Mycobacterium, Pseudomonas, and Streptomyces. It is well known from scientific and patent literature and from commercial practice that the Nocardioform-type cholesterol oxidase is distinct from the other microbial cholesterol oxidases.
The Nocardioform enzyme is very stable and active over a wide pH range (pH 6.0-8.0), the Km for cholesterol is 1.4.times.10.sup.-5 mol/liter at 25.degree., and the enzyme is highly specific for .DELTA..sup.4 - or .DELTA..sup.5 -3.beta.-sterols. As produced in the Nocardioforms, cholesterol oxidase is membrane associated and requires detergent extraction and lipid removal for purification.
Singer, et al., J. Bacteriol., 1988, Vol. 170, pp. 638-645, describes expression of genes in Rhodococcus sp. from both E. coli and Streptomyces sp. and the development of a shuttle plasmid based on an E. coli plasmid and containing a Rhodococcus plasmid-derived origin of replication. The plasmid is capable of replicating in E. coli and in several but not all of the Rhodococcus species tested.